scholarly journals Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

1981 ◽  
Vol 1 (11) ◽  
pp. 1048-1056 ◽  
Author(s):  
R C Dickson ◽  
R M Sheetz ◽  
L R Lacy
Keyword(s):  
Ribonucleic Acid ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Kluyveromyces Lactis ◽  
Mrna Level ◽  
Genetic Regulation ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Messenger Ribonucleic Acid ◽  
Beta Galactosidase

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid

1981 ◽  
Vol 1 (11) ◽  
pp. 1048-1056
Author(s):  
R C Dickson ◽  
R M Sheetz ◽  
L R Lacy
Keyword(s):  
Ribonucleic Acid ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Kluyveromyces Lactis ◽  
Mrna Level ◽  
Genetic Regulation ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Messenger Ribonucleic Acid ◽  
Beta Galactosidase

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

scholarly journals Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene.

1981 ◽  
Vol 1 (7) ◽  
pp. 629-634 ◽  
Author(s):  
L R Lacy ◽  
R C Dickson
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Regulation ◽  
Enzyme Induction ◽  
Ribonucleic Acid ◽  
Molecular Basis ◽  
Kluyveromyces Lactis ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Messenger Ribonucleic Acid ◽  
Beta Galactosidase

We examined the molecular basis for beta-D-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. The protein synthesis inhibitor anisomycin effectively blocked both protein synthesis and enzyme induction by lactose. Further, hybridization analysis with the cloned beta-galactosidase gene indicated coordinate increases in the concentration of beta-galactosidase messenger ribonucleic acid and enzyme activity. The half-life of beta-galactosidase messenger ribonucleic acid was the same (4.8 +/- 0.4 min) when measured both before and at succeeding times during enzyme induction. These results strongly support the hypothesis that expression of the yeast beta-galactosidase gene is subject to transcriptional regulation.

scholarly journals GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon.

1987 ◽  
Vol 7 (2) ◽  
pp. 780-786 ◽  
Author(s):  
M I Riley ◽  
J E Hopper ◽  
S A Johnston ◽  
R C Dickson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Fold Induction ◽  
Beta Galactosidase ◽  
Galactokinase Activity

A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.

Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene

1981 ◽  
Vol 1 (7) ◽  
pp. 629-634
Author(s):  
L R Lacy ◽  
R C Dickson
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Regulation ◽  
Enzyme Induction ◽  
Ribonucleic Acid ◽  
Molecular Basis ◽  
Kluyveromyces Lactis ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Messenger Ribonucleic Acid ◽  
Beta Galactosidase

We examined the molecular basis for beta-D-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. The protein synthesis inhibitor anisomycin effectively blocked both protein synthesis and enzyme induction by lactose. Further, hybridization analysis with the cloned beta-galactosidase gene indicated coordinate increases in the concentration of beta-galactosidase messenger ribonucleic acid and enzyme activity. The half-life of beta-galactosidase messenger ribonucleic acid was the same (4.8 +/- 0.4 min) when measured both before and at succeeding times during enzyme induction. These results strongly support the hypothesis that expression of the yeast beta-galactosidase gene is subject to transcriptional regulation.

GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon

1987 ◽  
Vol 7 (2) ◽  
pp. 780-786
Author(s):  
M I Riley ◽  
J E Hopper ◽  
S A Johnston ◽  
R C Dickson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Fold Induction ◽  
Beta Galactosidase ◽  
Galactokinase Activity

A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.

scholarly journals Activating Mutations of the Gsα Gene Are Associated with Low Levels of Gsα Protein in Growth Hormone-Secreting Tumors1

1998 ◽  
Vol 83 (12) ◽  
pp. 4386-4390 ◽  
Author(s):  
Emilia Ballaré ◽  
Simona Mantovani ◽  
Andrea Lania ◽  
Anna M. Di Blasio ◽  
Lucia Vallar ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Ribonucleic Acid ◽  
Pituitary Tumors ◽  
Acid Synthesis ◽  
Endocrine Tumors ◽  
Wild Type ◽  
Messenger Ribonucleic Acid ◽  
Activating Mutations ◽  
Gsα Gene ◽  
System Data

Evidence suggests the existence of a direct relationship between cellular Gsα content and activation of the adenylyl cyclase system. Data on Gsα levels in endocrine tumors that depend on cAMP for growth, particularly pituitary adenomas, are still limited. The levels of Gsα protein were evaluated in 11 GH-secreting adenomas with Gsα mutations (gsp+) and 15 without (gsp). Complementary DNAs from gsp+ tumors contained very low amounts of wild-type Gsα sequences, indicating a preponderance of the mutant Gsα transcripts in these tumors. Immunoblotting of Gsα protein showed that the two isoforms were present at high levels in all gsp−, but were undetectable or barely detectable in gsp+. The low Gsα content in gsp+ tumors was not due to a reduction in ribonucleic acid synthesis or stability, as Gsα messenger ribonucleic acid levels were similar in wild-type and mutant tissues. Treatment of gsp− cells with cholera toxin caused a marked reduction of Gsα levels. As in other cell systems cholera toxin increases Gsα degradation, our data are consistent with an accelerated removal of mutant Gsα. This may represent an additional mechanism of feedback response to the constitutive activation of cAMP signaling in pituitary tumors with mutations in the Gsα gene.

scholarly journals MUTATIONS AFFECTING SYNTHESIS OF β-GALACTOSIDASE ACTIVITY IN THE YEAST KLUYVEROMYCES LACTIS

Genetics ◽  
1980 ◽  
Vol 95 (4) ◽  
pp. 877-890
Author(s):  
R Michael Sheetz ◽  
Robert C Dickson
Keyword(s):  
Enzyme Activity ◽  
Kluyveromyces Lactis ◽  
Single Mutation ◽  
Chromosome 2 ◽  
Wild Type Allele ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Fold Level ◽  
Complementation Groups ◽  
Constitutive Synthesis

ABSTRACT Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac-) were isolated and characterized. All of the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3,lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal-). Since the Gal- and Lac- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate Chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.—To further characterize the nature of the Lac- phenotype, the basal and inducible level of β-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of β-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of β-galactosidase activity.

scholarly journals LAC4 IS THE STRUCTURAL GENE FOR β-GALACTOSIDASE IN KLUYVEROMYCES LACTIS

Genetics ◽  
1981 ◽  
Vol 98 (4) ◽  
pp. 729-745
Author(s):  
R Michael Sheetz ◽  
Robert C Dickson
Keyword(s):  
Thermal Stability ◽  
Structural Gene ◽  
Gel Electrophoresis ◽  
Kluyveromyces Lactis ◽  
Two Dimensional ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Nonsense Suppressor ◽  
Hydrolysis Of ◽  
Thermal Stability Of

ABSTRACT Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac+ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the Km for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is obs0ervable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac+ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.

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